Total DNA, RNA, and protein isolation - Macherey …

1 US:Tel.: +1 484 821 0984 Fax: +1 484 821 1272 E-mail: GmbH & Co. KG Neumann-Neander-Str. 6 8 52355 D ren / International:Tel.: +49 24 21 969-0 Fax: +49 24 21 969-199 E-mail: +41 62 388 55 00 Fax: +41 62 388 55 05 E-mail: +33 388 68 22 68 Fax: +33 388 51 76 88 E-mail: GmbH & Co. KG Neumann-Neander-Str. 6 8 52355 D ren / International:Tel.: +49 24 21 969-0 Fax: +49 24 21 969-199 E-Mail: +41 62 388 55 00 Fax: +41 62 388 55 05 E-Mail: +33 388 68 22 68 Fax: +33 388 51 76 88 E-Mail: ISO 9001 ZERTIFIZIERTMACHEREY-NAGEL GmbH & Co. KG Neumann-Neander-Str. 6 8 52355 D ren / International:Tel.: +49 24 21 969-0 Fax: +49 24 21 969-199 E-Mail: +41 62 388 55 00 Fax: +41 62 388 55 05 E-Mail: +33 388 68 22 68 Fax: +33 388 51 76 88 E-Mail: ISO 9001 ZERTIFIZIERTT otal DNA, RNA, and protein isolationUser manualNucleoSpin TriPrepApril 2018 / Rev. 06 Total DNA, RNA, and protein isolationProtocol at a glance (Rev.)

2 06) Macherey -NAGEL GmbH & Co. KG Neumann-Neander-Str. 6 8 52355 D ren GermanyTel.: +49 24 21 969-270 Fax: +49 24 21 969-199 TriPrep1 Homogenize sampleUp to 30 mg2 Lyse sample350 L RP1 L -mercaptoethanol (or comparable reducing agent)3 Filtrate lysate1 min, 11,000 x g4 Adjust DNA and RNA binding conditions350 L ethanol (70 %)5 Bind DNA and RNALoad sample30 s, 11,000 x gProtein Purification ( protein in the column flow-through)12 Precipitate protein10 700 L flowthrough1 vol PPRT, 10 min5 min, 11,000 x g13 Wash protein pellet500 L ethanol (50 %)1 min, 11,000 x g14 Dry protein pelletRT, 5 10 min15 Prepare protein sample20 100 L PSB-TCEP3 min, 95 98 C1 min, 11,000 x gDNA and RNA Purification (both bound to the silica membrane)6 Wash silica membrane1st and 2nd wash each:500 L DNA Wash1 min, 11,000 x g7 Dry mem-braneR T, 3 min (with open lid)8 Elute DNA100 L DNA EluteIncubate 1 min1 min, 11,000 x g9 Digest residual DNA95 L DNase reaction mixtureRT, 15 min10 Wash and dry silica membrane1st wash 2nd wash200 L RA2 600 L RA330 s, 11,000 x g3rd wash250 L RA32 min, 11,000 x g11 Elute highly pure RNA60 L H2O (RNase-free)1 min, 11,000 x g3 Macherey -NAGEL 04/2018, Rev.

3 06 Total DNA, RNA, and protein isolationTable of contents1 Components Kit contents Reagents, consumables, and equipment to be supplied by user About this user manual 52 Product description The basic principle Kit specifications Handling, preparation, and storage of starting materials Guideline for appropriate sample amount, precipitation volume, and resolubilization volume for protein isolation Elution procedures for DNA Elution procedures for RNA 133 Storage conditions and preparation of working solutions 144 Safety instructions 165 Protocols DNA, RNA, and protein purification from cultured cells and tissue Total RNA preparation from RNAlater treated samples rDNase digestion in solution 246 Appendix protein quantification Troubleshooting References Ordering information Product use restriction / warranty 39 Macherey -NAGEL 04/2018, Rev. 064 Total DNA, RNA, and protein isolation1 Components Kit contentsNucleoSpin TriPrepREF10 preps preps preps Buffer RP110 mL25 mL125 mLBuffer DNA Wash (Concentrate)*12 mL12 mL5 x 12 mLBuffer DNA mL12 mL3 x 12 mLWash Buffer RA215 mL15 mL80 mLWash Buffer RA3 (Concentrate)*6 mL12 mL3 x 25 mLRNase-free H2O13 mL13 mL60 mLProtein Precipitator PP9 mL45 mL225 mLProtein Solving Buffer PSB (without reducing agent) 2 x 1 mL40 mLReducing Agent TCEP2 x 14 mg107 mg5 x 107 mgReaction Buffer for rDNase7 mL7 mL30 mLrDNase, RNase-free (lyophilized)*1 vial (size C)1 vial (size D)5 vials (size D)NucleoSpin Filters (violet rings)1050250 NucleoSpin TriPrep Columns (light blue rings, plus Collection Tubes)1050250 Collection Tubes (2 mL)30150750 Collection Tubes ( mL)20100500 User manual111* For preparation of working solutions and storage conditions see section 04/2018, Rev.

4 06 Total DNA, RNA, and protein Reagents, consumables, and equipment to be supplied by userReagents 50 % ethanol (to prepare Buffer DNA Wash) 70 % ethanol (to adjust RNA binding conditions) 96 100 % ethanol (to prepare Wash Buffer RA3) Reducing agent ( -mercaptoethanol, or DTT (dithiothreithol), or TCEP (Tris(2-carboxyethyl) phosphine hydrochloride) to supplement lysis bufferConsumables mL microcentrifuge tubes (for sample lysis and DNA elution) Disposable RNase-free pipette tipsEquipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Thermal heating block Equipment for sample disruption and homogenization Personal protection equipment (lab coat, gloves, goggles)Additional material is furthermore needed for protein quantification, see section About this user manualIt is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin TriPrep kit is used for the first time.)

5 Experienced users, however, may refer to the Protocol at a glance instead. The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure. All technical literature is available on the internet at Please contact Technical Service regarding information about changes of the current user manual compared to previous 04/2018, Rev. 066 Total DNA, RNA, and protein isolation2 Product The basic principleIntroductionStudies of gene expression at the level of transcription and translation by quantification of RNA and protein combined with verification of genomic sequence ( , transgene integration site) are often hampered by the small sample size and the necessity of different often incompatible techniques for DNA, RNA, and protein isolation . Samples may comprise biopsies, tumors, tissues, transgene organisms, and others.

6 The NucleoSpin TriPrep kit enables isolation of DNA, RNA, and protein from diverse sample types. DNA, RNA, and protein are isolated without splitting the sample prior to extraction. Thus, DNA, RNA, and protein are obtained from one and the same sample and not from three similar portions of one sample. This is especially valuable for unique, small, and precious samples. DNA and RNA are eluted separately from the NucleoSpin TriPrep Column, with a low salt buffer and water, respectively. Isolated DNA and RNA are suitable for all common downstream applications. Isolated protein is suitable for SDS-PAGE, Western Blot analysis, and , RNA, and protein isolationOne of the most important aspects in the isolation of DNA, RNA, and protein is to prevent their degradation during the isolation procedure. With the NucleoSpin TriPrep method, cells are lysed by incubation in a solution containing large amounts of chaotropic ions.

7 This lysis buffer immediately inactivates virtually all enzymes ( , DNases, RNases, proteases, and phosphatases) which are present in almost all biological materials. The buffer dissolves even hardly soluble protein , creates appropriate binding conditions which favor adsorption of DNA and RNA to the silica membrane, and enables protein to pass the specially treated NucleoSpin TriPrep Column virtually quantitatively. Expensive and harmful proteinase inhibitors or inhibitor cocktails are not necessary due to the denaturing properties of the lysis buffer. After two special washing steps, DNA is eluted with a low salt buffer (DNA Elute) which selectively elutes DNA and keeps RNA quantitatively on the column. Eluted DNA is immediately ready for downstream applications without further purification. DNA elution prior to RNA elution does neither compromise RNA quantity nor quality. RNA isolated with the NucleoSpin TriPrep kit is of identical quality as RNA isolated with the well proven NucleoSpin RNA kit.

8 Residual DNA still bound to the silica membrane is removed by an rDNase solution which is directly applied onto the silica membrane during the preparation (RNase-free rDNase is supplied with the kit). Simple washing steps with two different buffers remove salts, metabolites, and macromolecular cellular components. Pure RNA is finally eluted under low ionic strength conditions with RNase-free water (supplied). protein is isolated from the column flowthrough. protein is precipitated in denatured form with a special buffer ( protein Precipitator PP) which effectively precipitates protein . After a washing step the protein pellet is dissolved in protein Solving Buffer (PSB) containing the odourless reducing agent TCEP. The protein can thus readily be applied to SDS-PAGE analysis. The kit is not recommended for isolation of native , RNA, and protein preparation using 1 kits can be performed at room temperature.

9 The DNA and RNA eluates, however, should be treated with care because RNA is very sensitive to trace contaminations of RNases, often found on general lab ware, fingerprints, and dust. DNA can be stored at 4 C for short term and at -20 C for long term storage. To ensure RNA stability keep RNA frozen at -20 C for short-term or -70 C for long-term storage. Recovered protein dissolved in protein Solving Buffer is unproblematic concerning 04/2018, Rev. 06 Total DNA, RNA, and protein Kit specifications NucleoSpin TriPrep kits are recommended for the isolation of Total DNA, RNA, and protein from cultured cells, tissue, and other biological 1: Kit specifications at a glanceParameterNucleoSpin TriPrepFormatMini spin columnSample material< 5 x 106 cultured cells*, < 30 mg human / animal tissue*, < 100 mg plant tissue* Total RNAT otal DNAT otal proteinFragment size> 200 b< 30 kbp15 300 kDaTypical yield< 70 g< 6 g< 1200 gA260 Typical RIN (RNA integrity number)> 9 Elution volume (RNA and DNA) Resolubilization volume ( protein )40 120 L100 L10 100 LPreparation time30 min/6 preps45 min/6 preps (RNA + DNA)35 min/6 prepsBinding capacity200 g10 g** DNA characteristics Isolated DNA is of high molecular weight and typically exceeds 20 kb.

10 DNA is commonly stable, even at 37 C for 2 h with or without addition of a typical restriction enzyme buffer, showing the absence of DNases. DNA is digestable with restriction enzymes and suitable for PCR.* For samples larger than approx. one million cells or 5 mg tissue, DNA yield does not increase lineary with the sample amount. DNA yield may decrease utilizing five million cells, 30 mg tissue, or more. Typically however, DNA yield is still sufficient for PCR analysis.** Binding capacity of DNA 10 g, strongly depending on RNA amount bound to the membraneMACHEREY-NAGEL 04/2018, Rev. 068 Total DNA, RNA, and protein isolationRNA characteristics The NucleoSpin TriPrep kit allows purification of pure RNA with an A260 / A280 ratio generally exceeding (measured in TE buffer, pH ). The isolated RNA is ready to use for applications like reverse transcriptase PCR (RT-PCR), primer extension, or RNase protection assays.